Studies of the Enzymic Capacities and Transport Properties of Pea Root Plastids

Plastids have been isolated from pea (Pisum sativum L.) roots with a high degree of purity and intactness. In these plastids, the activity of enzymes involved in carbohydrate metabolism have been analyzed and corrected for cytosolic contamination. The results show that fructose-1,6-bisphosphatase, NAD-glyceraldehyde phosphate dehydrogenase, and phosphoglyceromutase are not present in pea root plastids. Transport measurements revealed that inorganic phosphate, dihydroxyacetone phosphate (DHAP), 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, and glucose-6-phosphate (Glc6p) are transported across the envelope in a counterexchange mode. Transport of glucose-1-phosphate was definitely excluded. The oxidation of Glc6P by intact plastids resulted almost exclusively in the formation of DHAP. The parallel measurement of DHAP formation and NO2- consumption during Glc6P-supported nitrite reduction yielded a ratio of NO2-reduced/DHAP formed of 1.6, which is relatively close to the theoretical value of 2.0. These results show that the oxidation of Glc6P, involving the uptake of Glc6P and the release of DHAP, and the reduction of NO2- are very tightly coupled to each other.     

ADP/ATP Translocator from Pea Root Plastids (Comparison with Translocators from Spinach Chloroplasts and Pea Leaf Mitochondria)

The kinetic properties of the adenosine 5[prime]-diphosphate/adenosine 5[prime]-triphosphate (ADP/ATP) translocator from pea (Pisum sativum L.) root plastids were determined by silicone oil filtering centrifugation and compared with those of spinach (Spinacia oleracea L.) chloroplasts and pea leaf mitochondria. In addition, the ADP/ATP transporting activities from the above organelles were reconstituted into liposomes. The Km(ATP) value of the pea root ADP/ATP translocator was 10 [mu]M and that for ADP was 46 [mu]M. Corresponding values of the spinach ADP/ATP translocator were 25 [mu]M and 28 [mu]M, respectively. Comparable results were obtained for the reconstituted ATP transport activities. The transport was highly specific for ATP and ADP. Adenosine 5[prime]-monophosphate (AMP) caused only a slight inhibition and phosphoenolpyruvate and inorganic pyrophosphate caused no inhibition of ATP uptake. With pea root plastids and spinach chloroplasts, Km values >1 mM were obtained for ADP-glucose. Since the concentrations of ATP and ADP-glucose in the cytosolic compartment of spinach leaves have been determined as 2.5 and 0.6 mM, respectively, a transport of ADP-glucose by the ADP/ATP translocator does not appear to have any physiological significance in vivo. Although both the plastidial and the mitochondrial ADP/ATP translocators were inhibited to some extent by carboxyatractyloside, no immunological cross-reactivity was detected between the plastidial and the mitochondrial proteins. It seems probable that these proteins derive from different ancestors.     

The Utilization of Glycolytic Intermediates as Precursors for Fatty Acid Biosynthesis by Pea Root Plastids

Radiolabeled pyruvate, glucose, glucose-6-phosphate, acetate, and malate are all variously utilized for fatty acid and glycerolipid biosynthesis by isolated pea (Pisum sativum L.) root plastids. At the highest concentrations tested (3-5mM), the rates of incorporation of these precursors into fatty acids were 183, 154, 125, 99 and 57 nmol h-1 mg-1 protein, respectively. In all cases, cold pyruvate consistently caused the greatest reduction, whereas cold acetate consistently caused the least reduction, in the amounts of each of the other radioactive precursors utilized for fatty acid biosynthesis. Acetate incorporation into fatty acids was approximately 55% dependent on exogenously supplied reduced nucleotides (NADH and NADPH), whereas the utilization of the remaining precursors was only approximately 10 and 20% dependent on added NAD(P)H. In contrast, the utilization of all precursors was greatly dependent (85-95%) on exogenously supplied ATP. Palmitate, stearate, and oleate were the only fatty acids synthesized from radioactive precursors. Higher concentrations of each precursor caused increased proportions of oleate and decreased proportions of palmitate synthesized. Radioactive fatty acids from all precursors were incorporated into glycerolipids. The data presented indicate that the entire pathway from glucose, including glycolysis, to fatty acids and glycerolipids is operating in pea root plastids. This pathway can supply both carbon and reduced nucleotides required for fatty acid biosynthesis but only a small portion of the ATP required     

Characterization of Orthophosphate Absorption by Pea Root Protoplasts

Root protoplasts were isolated from 4 d old seedlings of Pisumsativum. Viability was verified by fluorescin diacetate, directblue and neutral red vital staining techniques. Phosphate influx of the protoplasts was similar to that of rootswith respect to phase 1 affinity for orthophosphate /{K_m = 99mmol m^–3) and sensitivity to pH and metabolic inhibitors.Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone decreaseduptake to 4% of the control and diethyl-stilboestrol inducedleakiness in the protoplasts. Influx was only slightly lessthan that in plasmolysed roots which in turn was 3.6 times lowerthan that of normal roots. Efflux from the protoplasts was highwith a value of approximately 2% of the cellular phosphate contentper hour. The elevated leakage indicated an essential differencebetween the effects of protoplast isolation on influx and effluxof phosphate. Key words: Root protoplasts, Phosphate absorption, Influx, Efflux, Metabolic inhibitors, Diethyl-stilboestrol, Subprotoplasts     

Characterization of Cyanobacterial Hydrocarbon Composition and Distribution of Biosynthetic Pathways

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.     

豌豆根胞质V1—ATPase的鉴定

利用免疫印迹,免疫电镜和ATP水解活性的测定对豌豆（Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定。用兔抗绿豆V-typeH^ -ATPase的A,B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A,B亚基。活性测定结果进一步表明胞质具有ATP水解活性,这些结果说明豌豆根胞质具有活性的V1-ATPase复合物。这是首次直接证明植物中有胞质V1-ATPase的存在。     

豌豆根胞质V1-ATPase的鉴定

利用免疫印迹、免疫电镜和ATP水解活性的测定对豌豆(Pisum sativum L.)根细胞胞质中V1-ATPase复合物的存在进行鉴定.用兔抗绿豆V-type H+-ATPase 的A、B亚基的抗体进行的immuno-blotting和胶体金电镜结果都表明,胞质中存在有A、B亚基.活性测定结果进一步表明胞质具有ATP水解活性.这些结果说明豌豆根胞质具有有活性的V1-ATPase复合物.这是首次直接证明植物中有胞质V1-ATPase的存在.     

Changes of Plastids During Xylem Differentiation in Barley Root

Plastids (eoplasts) are present in meristematic cells of prospectivecentral metaxylem in the barley root. Starch starts to be formedin plastids precisely after the cessation of mitotic activityand at the beginning of endomitotic growth. During secondarywall formation, the starch is gradually lost. Cavities are formedin plastids and signs of plastid degeneration are present fromthis stage of cell development. However, some intact globularplastids without starch are present until shortly before thefinal step of ontogeny, i.e. total destruction of protoplast. Hordeum distichum L., root, xylem, plastids, endomitotic growth, starch     

The Biosynthesis of Indole-3-acetic Acid from D-tryptophan in Alaska Pea Plastids

IAA biosynthesis in Alaska peas is shown to be plastid localized.D-tryptophan is a much better substrate than is L-tryptophan,and IAA production is dependent on a keto acid. In line withthis, a plastid localized D-tryptophan aminotransferase hasbeen found and purified 1,500 fold. The enzyme has no activitywith L-tryptophan and prefers pyruvic or oxaloacetic acid asan amino group acceptor. Activities are much higher in darkthan in light grown tissues. Some possible physiological ramificationsare discussed. (Received May 15, 1989; Accepted July 25, 1989)     

The Effect of Root Hypoxia and Iron Deficiency on the Photosynthesis, Biochemical Composition, and Structure of Pea Chloroplasts

The combined effect of root hypoxia and iron deficiency on biochemical composition, photosynthetic indices, and structure of pea (Pisum sativum L.) chloroplasts were investigated. Both factors suppressed chlorophyll accumulation and leaf photosynthetic activity, causing chlorosis. It was shown, that iron deficiency reduced more severe the light-harvesting complexes of photosystems (PS), and root hypoxia, the reaction center complexes of the photosystem I (PSI) and photosystem II (PSII). The combined action of both factors was stronger than the effect of each factor. However, even in yellow and almost white leaves, chloroplasts contained small amounts of all pigment–protein complexes and maintained weak photosynthetic activity, although their structure was poorly developed and comprised only vesicles and small thylakoids capable to form contacts and small grana. The conclusion is that the mechanisms of root hypoxia and iron deficiency destructive action are different and these factors differently and independently influenced leaf chloroplasts.     

Changes in the Biochemical Composition, Structure, and Function of Pea Leaf Chloroplasts in Iron Deficiency and Root Anoxia

A combined effect of iron deficiency and root anoxia on the biochemical composition, function, and structure of pea leaf chloroplasts was studied. It was found that the chlorosis of apical leaves in response to iron deficiency was determined by the reduction of light-harvesting complexes I and II. Under root anoxia, complexes of the reaction centers of photosystems I and II degraded first. Weak activity was preserved even in yellow and white leaves under the effect of both factors. The ultrastructure of leaf chloroplasts gradually degraded. Initially, intergranal thylakoid sites were reduced, and the longitudinal orientation of grana was disturbed. However, yellow and white leaves still retained small thylakoids and grana. It is concluded that the degrading effects of iron deficiency and root anoxia on the complex composition and leaf chloroplast structure and function are additive because of their autonomous mechanisms.     

Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings

Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-γ-glutamyl-d-alanine, and α-aminoadipic acid were exuded at different rates.     

Morphactin and Adventitious Root Formation in Pea Cuttings

Stock plants of pea (Pisum sativum L. cv. Alaska) were grown at different controlled levels of irradiance (4, 16 or 38 W m^?2) for 11 days from sowing. Morphactin (CFM, methyl-2-chloro-9-hydroxy-fluorene-9-carboxylate) was applied to the apex of the stock plants 3 days before cuttings were excised. The cuttings were rooted at 16 W m^?2. High levels of morphactin (>5 × 10^?3 mg l^?1) inhibited root formation in the cuttings. Low concentrations of CFM (5 × 10^?5 mg l^?1) promoted the formation of adventitious roots in cuttings from plants grown at all three levels of irradiance, with the most pronounced effect in cuttings from 4 W m^?2. Measurements of ethylene evolution by CFM-treated plants 3 days after application, revealed a stimulatory effect on ethylene production by high CFM concentrations.     

Hydrogen Uptake and Exchange by Pea Root Nodules

Hydrogenase activity in pea root nodules was studied by followinggas exchanges of hydrogen and deuterium. It was found that thenodules did not evolve hydrogen but that hydrogen was takenup when it was provided in the gas mixture. When increasingpartial pressures of deuterium were supplied, hydrogen was evolvedat a rate which increased as the pressure of deuterium increased.Deuterium was taken up at the same time as this hydrogen wasevolved. Hydrogen evolution in the presence of deuterium wasinhibited by nitrogen, while the uptake of deuterium remainedunaffected. It was concluded that pea root nodules have at leasttwo separate hydrogenase system that are working in oppositedirections and must thus be situated in sites of different oxidation-reductionpotentials within the nodule.     

Exogenous Hexoses Cause Quantitative Changes of Pigment and Glycerolipid Composition in Filamentous Cyanobacteria

Cyanobacteria Spirulina platensis and Nostoc linckia were grown in the presence of 5 mM and 50 mM glucose or 5 mM mannose, non-metabolisable glucose analogue that effectively triggers the repression of photosynthesis. Glucose evoked active cyanobacterial growth but chlorophyll (Chl) content decreased to some extent and porphyrins were excreted. The content of monogalactosyldiacylglycerol decreased in glucose-grown cyanobacteria and that of phosphatidylglycerol increased substantially. Mannose inhibited cyanobacteria growth as well as Chl synthesis, however, phosphatidylglycerol contents were higher than in respective control samples. In cyanobacterial cells glucose may not only inhibit photosynthetic processes, but also cause structural transformations of membranes which may be necessary for the activity of respiratory electron transport chain components under heterotrophic conditions.     

Purification and Characterization of Pea Epicotyl beta-Amylase

The most abundant β-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this β-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a β-amylase by end product analysis and by its inability to hydrolyze β-limit dextrin and to release dye from starch azure. Pea β-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K_m of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea β-amylase is competitively inhibited by its end product, maltose, with a K_i of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with α-cyclohexaamylose being a stronger inhibitor than β-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea β-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea β-amylase is the multichain type. Possible roles of pea β-amylase in cellular glucan metabolism are discussed.     

Development of Vacuolar Volume in the Root Tips of Pea

Cell and vacuole areas were measured by light microscopy inlongitudinal and transverse sections cut at 0.4-mm intervalsalong the apical 7.2 mm of the primary root of pea. The vacuolararea (or volume) fraction — that is, vacuole area (orvolume) divided by cell area (or volume) — increased fromabout 15 % in cells 0.4 mm from the distal boundary of the apicalmeristem (the cap /root junction), to about 85% in cells situated6.8–7.2 mm from that boundary. At each distance, vacuoledevelopment tended to be greater in the cortex than in the stele.Vacuoles occupied about 22% of the tissue volume in the first1 mm length of root (measured from the cap/root junction), about31 % of the tissue volume in the first 2 mm, and about 45% whensummed over the apical 5-mm length of root. Phosphorus supplyor deprivation produced only minor and non-significant changesin vacuole development. The results have implications affectingprevious estimates of cytoplasmic and vacuolar phosphate concentrationsin pea root tips. Pisum sativum L., pea, root, vacuole, volume     

Pigment Composition and Photosynthetic Activity of Pea Chlorophyll Mutants

Pea chlorophyll mutants chlorotica 2004 and 2014 have been studied. The mutants differ from the initial form (pea cultivar Torsdag) in stem and leaf color (light green in the mutant 2004 and yellow-green in the mutant 2014), relative chlorophyll content (approximately 80 and 50%, respectively), and the composition of carotenoids: the mutant 2004 contains a significantly smaller amount of carotene but accumulates more lutein and violaxanthin; in the mutant 2014, the contents of all carotenoids are decreased proportionally to the decrease in chlorophyll content. It is shown that the rates of CO₂ assimilation and oxygen production by mutant chlorotica 2004 and 2014 plants are reduced. The quantum efficiency of photosynthesis in the mutants is 29–30% lower than in the control plants; in their hybrids, however, it is 1.5–2 times higher. It is proposed that both the greater role of dark respiration in gas exchange and the reduced photosynthetic activity in chlorotica mutants are responsible for the decreased phytomass increment in these plants. On the basis of these results, the conclusion is drawn that mutations chlorotica 2004 and 2014 affect the genes controlling the formation and functioning of various components of the photosynthetic apparatus.